Field of the Invention
This invention relates to a novel method and novel device for separating and washing cells, particles, and molecules or analytes, unreacted reagents, and other materials of interest (MOI) from associated or contaminating material or unwanted material such as proteins, for use in many procedures such as assays, diagnostic procedures, and preparative processes.
It relates primarily to determination of antigens, antibodies and other proteins on blood cells, in blood serum and other bodily samples and the use of buffers and other liquids in the determination process wherein magnetic propulsion of magnetic particles through a clean wash fluid and onto a capture device is effected.
The invention further relates to blood banking immunological diagnostic testing and immunohematology and more particularly to blood cell serological testing using magnetic particles and magnets to separate bound entities to be measured from unbound entities.
More particularly still, the invention relates to the separation and washing of materials as aforesaid in conjunction with the determination of the presence or absence of a MOI in which red blood cells are used in the assay system through the use of magnetic forces to move relevant materials through a clean wash fluid zone thereby to effect a washing of the relevant materials, and into a novel capture device in which a capture agent is present.
The invention relates to blood bank assay systems, and with some modification also is applicable to cell separations and microscopy, immunoassays, genetic testing and molecular separations as well as many other scientific procedures and quality control processes, wherein magnetic particles and magnetic forces are used to effect the separation in a liquid medium.
Finally, it relates to performing the above procedures by utilizing magnetic forces on reactive magnetic particles and test liquids containing materials of interest.
Description of Related Art
Clinical and industrial laboratories and chemical manufacturing plants represent an enormous, widespread industry in which many procedures and processes require, or are significantly enhanced by separation of a known or unknown material of interest from materials in the process which are not of interest, and which may interfere with suitable performance of the procedure or processes.
Many of these procedures are assays performed to determine the presence or absence of the amount of a given entity in a sample which is present, along with many other entities. In the health and medical sectors, this includes, for example, the isolation, expansion and identification of genetic material, usually from body liquids or tissues, as well as the detection and quantification of antigens, antibodies, and other proteins and small molecules. On the environmental level, it is desirable to determine the amount or presence of materials in water, air, chemicals, foods and the like.
Modern-day immunoassays are a good example of the many and varied laboratory and manufacturing procedures and processes that require purification of a material by washing and separation from contaminants and other materials. Immunoassays in which small amounts of an analyte are sought and measured in a sample, have evolved from the early, generally chemical, formats of various kinds of techniques. Binding partners, either specific for a given epitope or polyclonal in nature, have been employed in reaction with the desired target (be it known or unknown) to produce an entity which can be detected through a label attached to it or through some discernable, measurable effect upon a component of the test or on a substrate reactable with the label. Generally speaking, most, of the commercially useful versions of such tests require that the reacted binding partner be separated from unreacted binding partner so as to determine whether the sought entity has reacted or not reacted and how much is present.
The need to separate reactants from interfering substances is a major design feature in immunoassays, blood bank procedures, and chemical synthesis procedures. Many variations on several separation or washing techniques have been utilized, often involving dilution with large quantities of wash solution or sample, and often presenting the element of the test which determines the ease, complexity, cost, overall time, and sensitivity of the test, as well as the design of automated assay systems and the volume of hazardous material to be controlled and discarded. Immunoassays typically use both dilution and decanting or lateral liquid flow on chromatographic membrane or paper strip or other flow-through device, with large quantities of diluent or sample as washing methods. If a convenient separation method could be made available, alternative methods would be more readily adopted. An appropriate alternative separation concept would be broadly useful for immunoassays, including the special case of immunoassays that are utilized in blood banking involving cellular antigens and antibodies to them, and sequential chemical synthetic procedures.
Historically, a very common wash method in immunoassays involves the immobilization of a reagent antibody on a portion of the surface of individually coated containers, such as a microtiter well or test tube, then washing away undesired material by repeated wash solution additions and decanting steps. When this method is used, the final reaction is observed in the same tube or well that was washed, with the possibility that any contaminating material attached to its sides or inadvertently remaining in the container would interfere with the specificity or sensitivity of the assay. Additionally, the discarded liquids are often contaminated with hazardous material. Similar problems are encountered when the assay is done on a chromatographic paper strip.
In general, washing involves one of several methods: passage of wash solution over an immobilized material, repeated addition and removal steps to remove by dilution either in conjunction with centrifugation or immobilization with magnetic forces, or moving material into dense fluids of varying specific gravities or stabilized gel by centrifugation or magnetic force. Typically, when stabilized gels or dense fluids are used they are directionally below the reaction mixture. It would be desirable to have a method that would bring the washed material into a more convenient position, above the reaction mixture, for further manipulation and have a greater range of specific gravity for the wash fluid so that wash fluid of greater, similar or lower specific gravity could be used in the same method.
Unfortunately, many of the washing systems typically involve repeated flushing and resuspension, centrifugation and decanting steps which are laborious when performed manually and greatly complicate automated diagnostic instruments and make them more expensive.
Washing by lateral flow, flow-through and similar methods require slower, less effective and dilutive washing steps where liquid or wash solution is moved over an immobilized material in a single step or repeated addition, dilution, and removal steps. These methods are cumbersome, requiring large amounts of wash solution which need to be disposed of as a hazardous material, and typically rely on washing the sample in the original container previously contaminated by the heterogeneous sample or reaction mixture.
In some laboratory methods the material of interest is specifically captured on particles or microspheres as the solid state surface. Such particles or microspheres are employed in many and varied diagnostic and preparative methods. Their primary purpose in these methods is to separate or purify specific items of interest from unwanted surrounding contaminating materials. Similarly, in blood bank red cell testing, the red cell itself is the particle surface that captures material of interest. The active binding agents affixing such particles include antibodies, ligands, lectins, oligonucleotides and many other specific binding molecules of non-immune origin. Methods are well known and commonly practiced which enable the preparation of particles and microspheres of various size, specific gravity, and other properties to be attached to reagents which can specifically bind to specific cells, viruses and sub-cellular particles or other materials of interest.
When particles of various kinds are bound to materials of interest, they become much larger complexes which settle more rapidly under Stokes law. Larger denser complexes can be sedimented or centrifuged from a mixture and thus washed. Unfortunately this method requires centrifugation to create force to move the particles, and the medium must have a lower specific gravity than the particles which restricts the media that can be used and limits the power of the separation.
Some current separation methods use, as an alternative to centrifugation, separation by a process in which magnetic particles are bound specifically to the materials of interest to form a complex which is then selectively separated from materials not of interest by the pull of a magnetic field rather than by centrifugation or by gravity alone. Usually the material to be separated is pulled to the side of the vessel by magnetic force and the material not of interest removed by decanting or rinsing or other liquid flow past the material of interest held on the wall.
A current art-preferred method of conducting a wide variety of assays involves the use of individually coated chromatographic strips whereby a sample suspected of containing the analyte sought to be determined is applied either alone, or with appropriate reagents, to a chromatographic membrane or layers of membranes and allowed by lateral flow to come into contact on the strip with previously immobilized materials. Depending on the nature of reactants chosen, the immobilized reagents act to separate the desired test components so that a proper determination of the presence of the analyte can be made. This procedure typically passes the sample and labeled reagents laterally along a chromatographic strip and into the binding zone to bind with an immobilized reagent. Non-specific binding material to the immobilized reagent or to the strip is to be avoided or eliminated and therefore sufficient wash liquid must pass through the zone to remove unbound material. Flow-through procedures work in similar fashion, however the sample and labeled reagents “flow-through” immobilized reagents held in vertical proximity, rather than the horizontal proximity used in the chromatographic approach.
While, in general, lateral flow methods have the advantage of eliminating centrifugation steps and much of the liquid handling steps required for washing reactants in other methods, many lateral flow methods involve reagent addition steps during the procedure. For example, Becton Dickinson ColorPac® lateral flow devices may require pipetting of as many as six reagents during an analytic procedure.
An alternative chromatographic strip technology has been described in U.S. Pat. Nos. 6,713,271 and 6,136,549 to Christopher C. Feistel. In these two patents, magnetic assay methods and systems are described in which uniform bulk-prepared microparticle reagents and liquid reagents are substituted for the immobilized materials commonly used on the strip. Instead, magnetic particle-tagged reagents participating in the test and flowing on the strip are captured and held at a desired site on the strip by a magnet field applied to the site. The captured particles are read to determine the presence or absence of the analyte sought. Large volumes of wash liquid are required to move sample and unbound reactants away from the observation zone.
Density gradient separation is a commonly used separation method that employs a density gradient column and centrifugation. Density gradient separation methods separate materials of a mixture based upon their density. Materials of different density will spin down under centrifugation until they reach a liquid media layer of equal specific gravity. They “float” and do not enter the regions of density equal or higher than their own.
In pre-transfusion testing (PTT) of patients and donors, the gel method is a reliable method of choice, and utilizes a centrifuge method of washing red cells that relies on spinning the cells through a fluid zone of carefully chosen specific gravity such that the red blood cells (RBC), will spin down and contaminating proteins will be floated off. This method was first used in the Simwash technique developed by Graham et.al. in the 1980s. Excellent washing is accomplished but a narrow specific gravity range is required
Separation based on the rate of sedimentation of particles through a density gradient to separate them from materials with a lower specific gravity that will either float on the density gradient or move to a different layer in the gradient or sediment at a slower rate than the larger particles has been used for blood bank serological testing. In this method, cells are forced by centrifugal force through a solution of intermediate density which allows the heavier red blood cells to pass through and floats the less dense serum on the top. The washed red cells can either be recovered following a decant step or be assayed in place by incorporation of a reagent into the solution of intermediate density, eliminating the need for a decant step.
Blood Banks collect more than 15 million units of blood annually for more than 14 million transfusions in the United States. Pretransfusion testing of patient and donor blood samples is an enormous industry distributed over nearly 10,000 large and small blood bank laboratories.
Blood Banks perform tests to determine the blood type of red blood cells of donors and patients, to detect antibodies in blood sera, and perform compatibility (crossmatch) tests and for potential infectious disease agents in every donor blood sample.
The following blood bank tests are among the most important and most frequently conducted tests:
Direct red cell antigen testing, typically ABO grouping and Rh typing
Reverse grouping (testing for antibodies in serum which react with A or B cells)
Antiglobulin based tests which require a serum protein removal step as a part of the procedure. These include indirect typing procedures for antigens (such as Kell, Duffy, Kidd and some Rh), direct antiglobulin test (test for serum proteins on an individuals' red cells), indirect antiglobulin tests (includes antibody screening, antibody identification and the crossmatch).
The following is a description of common blood bank reagents and techniques.
THE DIRECT COOMBS (Antiglobulin) TEST: The direct Coombs (antiglobulin) test is used to determine the in vivo bound incomplete antibody (IgG) bound to the red cells. It is also used in the investigation of anemias to demonstrate whether red blood cells are coated with incomplete antibody, including that of babies born to Rh-negative mothers. It will reveal whether antibodies have been adsorbed on the surface of the red cells while the baby was in the uterus and is important in diagnosing Rh hemolytic disease of the newborn. The direct Coombs (antiglobulin) test is performed by washing the red blood cells to be tested and attempting to agglutinate them with Coombs (antiglobulin) reagent. The Coombs reagent is widely available. This test, as well as the indirect test described below, are variously referred to herein as Coombs test, anti-globulin test, AHG test or variations thereof. The serum is variously referred to as Coombs serum, anti-human globulin serum, AHG serum or the like.
The antiglobulin test, associated with blood transfusion safety, is critically important and is the gold standard in pre-transfusion testing. The antiglobulin test detects whether red cell antibodies have reacted with red blood cells. Current test methods require careful washing of cells by multiple centrifugation steps before the test can be run and read. Because of concern for potential neutralization of Coombs serum, a reagent used for antiglobulin testing, much care in washing and agglutination reading skill is needed in manual antiglobulin method.
THE INDIRECT COOMBS (Antiglobulin) TEST: The indirect Coombs (antiglobulin) test is used to screen the patient's serum for atypical antibodies such as Rho (D), Kell (K), Duffy (Fya), and hr′ (c) with known antigens. The presence of any of these atypical antibodies can cause hemolytic disease of the newborn or transfusion reactions.
In the indirect test, an unknown serum is tested with human group O reagent red blood cells. Group O reagent antibody screening cells are available commercially. They are a group of two or three O Rh positive and Rh negative donor red blood cells selected so as to be positive on at least 50% of the cells for each of the common clinically important red blood cell antigens. If a serum gives a positive reaction with such screening cells, tested separately or as a mixture, it must contain an atypical antibody of unknown identity. The techniques involved in performing the direct and indirect antiglobulin and the reasons therefor, are well-known in the art.
ABO GROUPING: Red cell (forward) typing with anti-A or anti-B reagents will demonstrate the presence or absence of A and B antigens on the red cell. Serum (reverse) typing with reagent A and B red cells will demonstrate the presence of anti-A and anti-B in the serum.
OTHER REAGENTS USEFUL IN ABO GROUPING: Other reagents may be used routinely in ABO grouping. They are often essential for resolving discrepancies between forward and reverse typing. Blood is not usually released from the blood bank for transfusion until any such discrepancies have been resolved. Anti-A, B (Group O serum) can detect weak A variants that may be missed by regular anti-A reagent. Other reagents include Anti-A1 reagent (absorbed B serum or Dolichos lectin), Anti-H lectin (Ulex), Reagent O Rh-positive screening cells, Reagent A2 cells and others.
COMPATIBILITY TESTING: Crossmatch (compatibility) tests are performed to determine the suitability of the donor's blood for the particular recipient. Blood transfusions are not given before performing a major crossmatch to test the donor's red cells against the serum of the recipient. If both donor and recipient are of the same blood group, a minor crossmatch may be done to test the recipient's red cells against the donor's serum. The minor crossmatch is of no value when donor and recipient belong to different blood groups because agglutination will occur. Major Crossmatch involves mixing donor's red cells with recipient's serum, incubating, centrifuging and adding antiglobulin reagent. Minor Crossmatch includes mixing donor's serum with recipient's red cells, incubating, centrifuging and adding antiglobulin reagent.
Rh TYPING: The crossmatch makes it possible to avoid hemolytic transfusion reactions following a particular transfusion. Blood banks are also concerned about isosensitization. If, for example, a blood bank selects Rho (D)-positive blood for an Rho (D)-negative woman, she will not have an incompatible crossmatch or a transfusion reaction if she has no anti-Rho (D) antibodies in her blood, but she may become sensitized to the Rho (D) antigen. Initiation of the immune response presents problems for subsequent transfusions and for subsequent pregnancies if she has an Rho (D)-positive mate. Rho (D) negative donors, Rho (D)-negative women and their Rho (D)-negative mates, and Rho (D)-negative cord bloods are tested for the presence of Rho\variant (DU) antigen that may not always be detected without the antiglobulin test. Various Rh typing methods and the appropriate controls are well-known to the art.
ANTIBODY TESTS: Screening for antibodies is especially important for patients receiving blood and the obstetrical patient. In obstetrical patients, early detection allows time to prepare for possible intrauterine or exchange transfusion in cases of Rh hemolytic disease of the newborn. Once the presence of an antibody has been detected, the problem of its identification remains, but this has been simplified by the development of antibody identification panels of group O reagent red cells. These screening and identification methods are well known to those skilled in the art.
Most blood bank tests require a wash step during the procedure. The centrifugal washing step, either by dilution and decant or sedimentation into beads or gel, takes about 5 to 10 minutes. The indirect antiglobulin test (IAT) is the most used and most reliable test in blood banking to determine binding of antibodies to red blood cells. This test is performed manually in test tube, requires addition of red cells and antisera, three manual centrifugation and decanting steps and finally a careful evaluation by a skilled technologist of whether the red cells have agglutinated, and recording of results. It is very labor intensive.
Although red cells in the presence of appropriate antibodies may clump in the absence of centrifugal forces, centrifugal procedures are typically used for almost all blood bank serological assays to cause enhanced aggregation of red cells for naked eye visualization at the assay end point. This is a major cause of a need to repeat an assay.
Classically, blood bank methods for determining blood types or detecting red cell antibodies in donor or patient sera are done manually in a large percentage of blood banks and rely upon hemagglutination as the endpoint to determining whether red cell antibodies have reacted with red blood cells in donor or patient blood samples.
Blood bank testing procedures have historically been a somewhat special case in the immunosassay art because the red blood cell, which is not visible to the naked eye, can form agglutinates that are visible to the naked eye and have a pattern distinguishable from that of nonaggregated red blood cells. Thus the typical blood bank procedure relies on human pattern recognition to detect a reaction. In blood bank testing, a wide variety of tests are performed using traditional, manual techniques that have been used for decades.
In recent years the manual DiaMed-ID (D-ID) antiglobulin gel test (Ortho Clinical Diagnostics, Raritan, N.J.) has largely replaced the classical manual method. It requires a ten minute centrifugation step and a more straightforward manual reading.
More and more testing is now being performed on automated instruments. For example, the Ortho Pro-vue (Ortho Clinical Diagnostics, Raritan, N.J.) is an automatic gel technology system, while the NEO and Galileo instruments (Immucor, Norcross, Ga., TANGO Optimo (Bio-Rad, Cressier-sur-Morat, Switzerland) and the Beckman PK7300 blood center instruments (Beckman Coulter, Brea, Calif.) are other large volume systems. The Immucor and Bio-Rad instruments require centrifugation while the Beckman instrument is not able to perform the antigolublin test.